Comprehensive two-dimensional gas-chromatographic analysis, also referred to as GC×GC (or else as “comprehensive 2D GC”), is a recent technique of analysis that involves subjecting the sample to be analysed to a first separation in a traditional capillary column and then injecting all the gaseous effluents of the first column, appropriately focused (concentrated) in fractions of predefined size, into a second capillary column having characteristics, and hence capacity of analytical detection, different from the first capillary column.
GC×GC analysis enables, thanks to fractionation (i.e. splitting in fractions) of the effluents of the first column and to the subsequent analysis of the fractions in the second column, a high analytical resolution that leads to a better and more sensitive identification of the substances present in the sample analysed.
In greater detail, the method of GC×GC analysis leads to a separation of the sample in a first capillary column and a further separation of the effluents of the first column, appropriately modulated, in a second column set in series to the first. All the effluents of the first column are subjected to a periodic modulation in time that consists in their subdivision and focusing in adjacent fractions having a constant extension in time by means of modulators (for example, thermal modulators) in which the effluents from the first column are first slowed down (focusing) and then accelerated again for introduction into the second column. Each fraction of the effluents thus modulated, i.e., each gas pulse, is then sent into the second capillary column so as to be further separated and analysed by a detector set in a position corresponding to the output section of the said second capillary column.
To guarantee the continuity of the analysis, the second capillary column is a column of a fast type, i.e., shorter and with a smaller diameter than the first capillary column, and the focused fractions of the effluents from the first column have extremely small time dimensions (<100 ms), thanks to the choice of an appropriate frequency of modulation of the said effluents.
The results of GC×GC analysis may be viewed by means of a three-dimensional Cartesian chromatogram, or by means of a contour plot (i.e. a level-curve graph), that presents two temporal axes (or time dimensions), one for each dimension of the analysis, and one axis (or level curve) along which there appears the intensity of the signal at output from the detector downstream of the second column. The peaks in the chromatogram, the height of which is proportional to the intensity of the signal at output from the corresponding detector and the position of which along each axis is a function of the instant of outflow (and hence of volatility) from the column considered of the substance or mixture at output, indicate the presence of a certain substance or mixture of substances.
In particular, the chromatogram is obtained as follows: each effluent, generated by the separation of the sample, can be detected, together with the instant of outflow, at output from the first capillary column, giving rise to a peak of height proportional to its quantity. This technique is the one usually adopted in one-dimensional gas chromatography. The effluents from the first column are then modulated in adjacent fractions, as described above, and each fraction is sent into the second capillary column, where it undergoes a further separation. Downstream of the second capillary column, the effluents from the latter are detected, together with the instant of output, giving rise to a series of peaks of restricted dimensions proportional to the size of each substance or mixture further separated in the second column.
The detection of the peaks at output from the second capillary column, which is obtained via discrete sampling, forms a continuous time series of digital signals in time proportional to the effluents from the second capillary column. To create the chromatogram, the continuous series of digital signals must be subdivided into a plurality of subsets (“cuts” or bands) of constant time dimension (and hence of equal number given by the discrete acquisition of the peaks), in such a way that their ordering side-by-side will give rise to a matrix in which one axis indicates the number of signals (points) acquired of the chromatogram of the second dimension and the other axis indicates the number of chromatograms of the second dimension.
Given that the number of bands (or subdivisions) of the second dimension, corresponding, that is, to the second capillary column, must be equal to the number of fractions of the effluents from the first column, the dimension of each subset, and hence the time interval of each subdivision, must therefore theoretically have the same time dimension as each fraction modulated at output from the first capillary column.
In other words, the amplitude of each modulation interval should theoretically coincide with the amplitude of each subset that divides the continuous time series of digital signals acquired.
However, the periodic modulation of the effluents from the first column takes place with a frequency that is determined beforehand on the basis of the characteristics of the first and second columns, whilst the acquisition by discrete points (sampling) of the peaks detectable downstream of the second column is obtained with a frequency of its own of the detector.
This means that the number of points, which are equidistant in time, acquired by the detector downstream of the second capillary column cannot be, and usually is not, perfectly contained in the time interval of modulation of the effluents from the first column, and this leads to a time drift in the acquisition of these points. If, for example, the time interval of each modulation is equal to 50 ms (20 Hz) and the acquisition of the sampling points is obtained every 0.3 ms (3.33 MHz), the number of sampling points in each modulation interval will be equal to 166, but there will be a time deviation equal to 0.2 ms, which will lead to an offset (out-of-phase) equal to 0.1 ms in the acquisition of the first sampling point and of the 166th sampling point of the subsequent modulation interval and which, in the third subsequent modulation interval, will lead to an offset in the start of sampling, whereby the 166th sampling point will coincide with the instant of start of the fourth modulation interval, and thus a sampling point will be lost in the third interval and so forth (the so-called “leap-year effect”). This situation, which is linked to the offset between the intervals of modulation and acquisition of the data downstream of the second capillary column, generates a time drift in the data acquired, and hence in the positions of the peaks detected in time that frequently renders any interpretation thereof difficult, if not indeed impossible.
An example of this time drift in the sampling is given in FIG. 1b, in which the axis of the sampling times is inclined as a result of the offset referred to above.
The U.S. Pat. No. 5,135,549 in the name of Phillips and Liu proposes synchronizing the start of the modulation of the effluents from the first column with the start of the acquisition of the data, when the detector downstream of the second column is of the scanning type, such as, for example, a mass spectrometer. This technique, albeit reiterated at each modulation pulse of the sample, does not solve the problem of the offset between acquisition frequency and modulation frequency and furthermore presupposes that the detection device presents an initial transient zero and will enable a frequent re-initialization of the detection.
A purpose of the present invention is to provide a method for detecting substances or mixtures of substances within a sample in an apparatus for comprehensive two-dimensional gas-chromatographic analysis (GC×GC) that does not present the drawbacks of the known art and that in particular will prevent the time drift of the data acquired on the corresponding chromatogram.
Another purpose of the present invention consists in providing a method of GC×GC analysis that will prove particularly precise and that does not require a continuous synchronization of the modulator and of the detector.
A further purpose of the present invention is that of providing an apparatus for comprehensive two-dimensional gas-chromatographic analysis (GC×GC) of the type comprising a first separation column, a second separation column, and a modulator of the effluents from the first separation column, which will yield chromatograms that are free from time drift and are extremely precise.